Stability of ten serum tumor markers after one year of storage at -18°C.

OBJECTIVES: The storage of serum tumor markers (STM) at -18 °C for one year has been a legal requirement in France since 1999, but has been abolished in 2022. This raises the question of the relevance of maintaining these biobanks in terms of conditions of storage. These should only be implemented after validation; in order to maintain the integrity of the biological sample and must be controlled over time according to the laboratory's procedures. The aim of the study was to assess the suitability of storing 10 STMs by evaluating their stability after one year of storage at -18 °C. METHODS: A new immuno-enzymatic assay (A+1) was conducted on samples that had been stored at -18 °C for one year after an initial assay (A) of one of the following STMs: carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), total (TPSA), and free (FPSA) prostate-specific antigen, calcitonin (CT), thyroglobulin (TG), and neuro-specific enolase (NSE). The results were confronted to four different permissible error sources. RESULTS: In total, 1148 A+1 assays were performed. A strong correlation between A+1 and A values was found for all analytes, but with a statistically significant reduction in the mean A+1 concentration compared to the mean A concentration in 7/10 STMs. The bias induced by conservation seems to be technically unsustainable if we rely on the repositories closest to the current analytical performances. CONCLUSIONS: These results support the discontinuation of mandatory STM biobank storage at -18 °C, which requires considerable technical time and organizational effort.

Discovering the ANK2-related autism phenotype.

Interestingly, disease-causing mutations in the ANK2 gene have been identified in patients with autism since 2012, though with no full clinical description. In this Research Letter, for the first time, we describe the detailed characteristics of a patient with autism caused by a new mutation in this gene. Our report is a first step to better understanding ANK2-related autism and will contribute to facilitating its further diagnosis.

A new approach to assessing calcium status via a machine learning algorithm.

BACKGROUND AND AIMS: Calcium plays a fundamental role in biological processes. Ionized calcium (Ca2+), is the biologically active fraction, but in practice total or corrected calcium assays are routinely used to determine calcium status. MATERIALS AND METHODS: We retrospectively compared total and corrected calcium to assess the calcium status, with ionized calcium which is considered for now like the best indicator. To compensate for their lack of performance we created a machine learning algorithm to predict calcium status. RESULTS: Corrected calcium performed less well than total calcium with 58% and 74% agreement, respectively, in our population. Total calcium was especially good for hypocalcemic samples: 93% agreement versus 45% for normocalcemic and 54% for hypercalcemic samples. Corrected calcium was especially good for hypercalcemic and normocalcemic samples: 90% and 84% agreement respectively versus 40% for hypocalcemic samples. Corrected calcium is mainly faulty in hypoalbuminemia, acid-base disorders, renal insufficiency, hyperphosphatemia, or inflammatory syndrome. With our ML algorithm, we obtained 81% correct classifications. Its main advantage is that its performance are not influenced by the variables studied or the calcium status. CONCLUSION: In many situations, corrected calcium should not be used. Our ML algorithm may make a better assessment of calcium status than total calcium. Finally, if doubt, an ionized calcium assay should be performed.

Genetic variations related to inflammation in suicidal ideation and behavior: A systematic review.

Background/objectives: Immune-inflammatory changes have been found in all types of suicidal ideation and behavior (SIB), independently of associated mental disorders. Since several Single Nucleotide Polymorphisms (SNPs) affect the function of inflammation-related genes, we searched the literature for genetic variations potentially altering inflammatory processes in SIB. Methods: We included studies that looked for associations between SIB and SNPs in genes related to inflammatory processes. Case reports, literature reviews, and animal studies were excluded. Articles were retrieved from PubMed and PsycINFO databases, Google Scholar and GreySource Index until September 17th, 2022. Quality was assessed using Q-Genie. Results: We analyzed 32 studies. SIB has been associated with eighteen SNPs located in genes encoding for interleukin-8 (rs4073), C-reactive protein (rs1130864), tumor necrosis factor α (rs1800629, rs361525, and rs1099724), tumor necrosis factor receptor 2 (rs1061622), transforming growth factor ß-1 (rs1982073), acid phosphatase 1 (rs7419262, rs300774), interleukin-10 (rs1800896), interferon γ (rs2430561), amino-carboxy muconate semialdehyde decarboxylase (rs2121337), interleukin 7 (rs10448044, rs10448042), macrophage migration inhibitory factor (rs755622), interleukin 1-α (rs1800587), and interleukin 1-ß (rs1143634 and rs16944. A genome-wide association study reported one association at the threshold of significance with the rs300774 SNP, located in the 2p25 region containing ACP1 gene. Discussion: The studies included were methodologically and clinically diverse and of moderate quality. Their findings suggest that some inflammation-related SNPs could increase the likelihood of SIB but the evidence to date is insufficient. Further research using gene-gene (GxG) and gene-environment (GxE) approaches is warranted. Systematic review registration: [https://www.crd.york.ac.uk], identifier [CRD42022296310].

Compound heterozygous P67S/D91A SOD1 mutations in an ALS family with apparently sporadic case.

Objectives: To describe a family with heterozygous P67S and D91A SOD1 mutations. Methods: The ALS profile of the proband was described. SOD1 gene sequencing was performed in the proband and his children. Results: The affected individual presented with progressive left peripheral facial palsy and slow progression with late limb involvement. Unequivocal upper and lower motor neuron signs were present, together with diffuse denervation at myography. The absence of trigeminal involvement excluded a FOSMN syndrome. Pedigree analysis did not show any other ALS case in the family. Genetic analysis of this patient showed P67S and D91A SOD1 mutations. The genetic analysis of the children showed that the mutations were each one carried by a different chromosome. Conclusions: P67S SOD1 mutation has been described in several ALS cases, either with familial or apparently sporadic ALS. The mutation is located in a mutational hotspot and was predicted pathogenic by in silico prediction software. The study of phylogenetic data show that at this codon, the proline is highly conserved throughout species reinforcing causality. Conversely, the D91A variant is known to have a recessive influence. Unilateral motor facial involvement, even after several years, in an ALS patient is unusual. The present case with compound heterozygosity and unusual onset in a patient with apparently sporadic ALS, widens the clinical spectrum of the disease and adds further arguments to support the systematic genetic screening of all ALS cases in referral ALS clinics.

Evaluation of CSF1R-related adult onset leukoencephalopathy with axonal spheroids and pigmented glia diagnostic criteria.

BACKGROUND AND PURPOSE: Diagnostic criteria for adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) due to colony-stimulating factor 1 receptor (CSF1R) mutation have recently been proposed. Our objective was to assess their accuracy in an independent multicenter cohort. METHODS: We evaluated the sensitivity and specificity of the diagnostic criteria for ALSP (including the "probable" and "possible" definitions) in a national cohort of 22 patients with CSF1R mutation, and 59 patients with an alternative diagnosis of adult onset inherited leukoencephalopathy. RESULTS: Overall, the sensitivity of the diagnostic criteria for ALSP was 82%, including nine of 22 patients diagnosed as probable and nine of 22 diagnosed as possible. Twenty of the 59 CSF1R mutation-negative leukoencephalopathies fulfilled the diagnostic criteria, leading to a specificity of 66%. CONCLUSIONS: Diagnostic criteria for ALSP have an overall limited sensitivity along with a modest specificity. We suggest that in patients suspected of genetic leukoencephalopathy, a comprehensive magnetic resonance imaging pattern-based approach is warranted, together with white matter gene panel or whole exome sequencing.

Juvenile amyotrophic lateral sclerosis associated with biallelic c.757delG mutation of sorbitol dehydrogenase gene.

Mutation in the sorbitol dehydrogenase gene (SORD) has been recently described to cause axonal Charcot-Marie-Tooth disease (CMT), intermediate CMT, and distal hereditary motor neuropathy (dHMN). We herein report the case of a 24-year-old patient diagnosed with juvenile amyotrophic lateral sclerosis (JALS) who carried the homozygous c.757delG mutation in SORD. No other pathogenic variant in frequent JALS-causative genes was found. Our findings expand the phenotype related to SORD mutation, a new and potentially treatable genetic disease.

Amyotrophic lateral sclerosis associated with a pathological expansion in the ATXN7 gene.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant hereditary neurodegenerative disease caused by the expansion of a CAG-repeat in the ataxin-7 (ATXN7) gene, usually characterized by progressive cerebellar ataxia and retinal dystrophy. We report the case of a 45-year-old woman presenting with a rapid-onset amyotrophic lateral sclerosis (ALS) phenotype associated with a 39-CAG-repeat expansion in ATXN7. This patient had neither ataxia nor retinal dystrophy, but she had an oculomotor cerebellar syndrome and a family history suggestive of SCA7. In SCA7, shorter expansions may be associated with less severe and incomplete clinical phenotypes, which could explain the patient's phenotype. Unknown genetic and environmental factors may also influence the patient's phenotype. We suggest that a pathological expansion in ATXN7 should be considered in cases of ALS-like phenotype, particularly when associated with oculomotor abnormalities or a family history of ataxia or blindness.

A recurrent SHANK3 frameshift variant in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is genetically complex with ~100 copy number variants and genes involved. To try to establish more definitive genotype and phenotype correlations in ASD, we searched genome sequence data, and the literature, for recurrent predicted damaging sequence-level variants affecting single genes. We identified 18 individuals from 16 unrelated families carrying a heterozygous guanine duplication (c.3679dup; p.Ala1227Glyfs*69) occurring within a string of 8 guanines (genomic location [hg38]g.50,721,512dup) affecting SHANK3, a prototypical ASD gene (0.08% of ASD-affected individuals carried the predicted p.Ala1227Glyfs*69 frameshift variant). Most probands carried de novo mutations, but five individuals in three families inherited it through somatic mosaicism. We scrutinized the phenotype of p.Ala1227Glyfs*69 carriers, and while everyone (17/17) formally tested for ASD carried a diagnosis, there was the variable expression of core ASD features both within and between families. Defining such recurrent mutational mechanisms underlying an ASD outcome is important for genetic counseling and early intervention.

Impact of a frequent nearsplice SOD1 variant in amyotrophic lateral sclerosis: optimising SOD1 genetic screening for gene therapy opportunities.

OBJECTIVE: Mutations in superoxide dismutase 1 gene (SOD1), encoding copper/zinc superoxide dismutase protein, are the second most frequent high penetrant genetic cause for amyotrophic lateral sclerosis (ALS) motor neuron disease in populations of European descent. More than 200 missense variants are reported along the SOD1 protein. To limit the production of these aberrant and deleterious SOD1 species, antisense oligonucleotide approaches have recently emerged and showed promising effects in clinical trials. To offer the possibility to any patient with SOD1-ALS to benefit of such a gene therapy, it is necessary to ascertain whether any variant of unknown significance (VUS), detected for example in SOD1 non-coding sequences, is pathogenic. METHODS: We analysed SOD1 mutation distribution after SOD1 sequencing in a large cohort of 470 French familial ALS (fALS) index cases. RESULTS: We identified a total of 27 SOD1 variants in 38 families including two SOD1 variants located in nearsplice or intronic regions of the gene. The pathogenicity of the c.358-10T>G nearsplice SOD1 variant was corroborated based on its high frequency (as the second most frequent SOD1 variant) in French fALS, the segregation analysis confirmed in eight affected members of a large pedigree, the typical SOD1-related phenotype observed (with lower limb onset and prominent lower motor neuron involvement), and findings on postmortem tissues showing SOD1 misaccumulation. CONCLUSIONS: Our results highlighted nearsplice/intronic mutations in SOD1 are responsible for a significant portion of French fALS and suggested the systematic analysis of the SOD1 mRNA sequence could become the method of choice for SOD1 screening, not to miss these specific cases.

Premature termination codons in SOD1 causing Amyotrophic Lateral Sclerosis are predicted to escape the nonsense-mediated mRNA decay.

Amyotrophic lateral sclerosis (ALS) is the most common and severe adult-onset motoneuron disease and has currently no effective therapy. Approximately 20% of familial ALS cases are caused by dominantly-inherited mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), which represents one of the most frequent genetic cause of ALS. Despite the overwhelming majority of ALS-causing missense mutations in SOD1, a minority of premature termination codons (PTCs) have been identified. mRNA harboring PTCs are known to be rapidly degraded by nonsense-mediated mRNA decay (NMD), which limits the production of truncated proteins. The rules of NMD surveillance varying with PTC location in mRNA, we analyzed the localization of PTCs in SOD1 mRNA to evaluate whether or not those PTCs can be triggered to degradation by the NMD pathway. Our study shows that all pathogenic PTCs described in SOD1 so far can theoretically escape the NMD, resulting in the production of truncated protein. This finding supports the hypothesis that haploinsufficiency is not an underlying mechanism of SOD1 mutant-associated ALS and suggests that PTCs found in the regions that trigger NMD are not pathogenic. Such a consideration is particularly important since the availability of SOD1 antisense strategies, in view of variant treatment assignment.

Clinical and Molecular Landscape of ALS Patients with SOD1 Mutations: Novel Pathogenic Variants and Novel Phenotypes. A Single ALS Center Study.

Mutations in the copper zinc superoxide dismutase 1 (SOD1) gene are the second most frequent cause of familial amyotrophic lateral sclerosis (ALS). Nearly 200 mutations of this gene have been described so far. We report all SOD1 pathogenic variants identified in patients followed in the single ALS center of Lyon, France, between 2010 and 2020. Twelve patients from 11 unrelated families are described, including two families with the not yet described H81Y and D126N mutations. Splice site mutations were detected in two families. We discuss implications concerning genetic screening of SOD1 gene in familial and sporadic ALS.

Clinical Phenotype and Inheritance in Patients With C9ORF72 Hexanucleotide Repeat Expansion: Results From a Large French Cohort.

BACKGROUND: In familial amyotrophic lateral sclerosis (ALS) cases, the presence of an abnormal C9ORF72 repeat expansion (C9RE) is the most frequent genetic cause identified. Various clinical phenotypes have been described in relation to the presence of C9RE, including psychiatric disorders or Huntington-like symptoms. In a subset of sporadic ALS, C9RE has also been described. In the present study, all index cases with ALS and C9RE identified in our center and their clinical profile, as well as neurological and psychiatric characteristics of identified family members, were described. Clinical characteristics of ALS patients were compared to 999 patients with sporadic ALS (SALS) from our database. RESULTS: From the 70 index cases with ALS identified, a total of 200 individuals were studied, 118 with ALS, 32 with fronto-temporal lobe degeneration (FTD), 37 with ALS/FTD, and 13 with psychiatric disorders. A familial history was present in 57 of the index cases (81%). In ALS and ALS/FTD cases with C9RE, the age of onset (AoO) was earlier than that in SALS cases, p < 0.0001 and p = 0.008, respectively. Sporadic cases with C9REALS (n = 13) had an earlier AoO compared to familial C9REALS ones, p < 0.0001. Within families, there was an earlier AoO in index cases and their siblings compared to their parental generation (p < 0.01). There was also a significant intrafamilial correlation for bulbar onset of ALS. The parental generation had significant female predominance compared to index cases and their siblings (sex ratio 0.47 vs. 1.4, p = 0.004), and this predominance was also present when considering parent-child pairs. In the group with psychiatric disorders, suicide was prominent (n = 9) and mean age was 54 years. CONCLUSION: Although our sample size is rather limited, the earlier AoO in index cases and their siblings compared to the parental generation may suggest an anticipation. Reasons for predominance of female transmission are unclear, but the hypothesis that gender influences transmission of the genetic trait or C9RE size variation may be taken into account. Intrafamilial correlation suggests that genetic aspects underlie the occurrence of bulbar onset in ALS patients. Studies on larger samples are warranted to confirm those results.

Spinal Motoneuron TMEM16F Acts at C-boutons to Modulate Motor Resistance and Contributes to ALS Pathogenesis.

Neuronal Ca2+ entry elicited by electrical activity contributes to information coding via activation of K+ and Cl- channels. While Ca2+-dependent K+ channels have been extensively studied, the molecular identity and role of Ca2+-activated Cl- channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca2+-activated Cl- conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS.

Theme 4 In vivo experimental models.

Background: In 90% of Amyotrophic Lateral Sclerosis (ALS) cases, the disease is sporadic, the remaining 10% being familial. Many genes have been associated with the disease. The use of next generation sequencing has allowed increasing the number of genes analysed in routine diagnostics. However, this increase raises the issue of genetic variants interpretation within a growing number of ALS-associated-genes. Variant classification is based on a combinatory analysis of multiple factors. Among them, functional analyses provide strong arguments on pathogenicity interpretation.Objectives: We developed a simple animal model, the Zebrafish, for the functional analysis of candidate variants pathogenicity identified by routine genetic testing.Methods: Transient overexpression of different ALS associated genetic variants has been performed by mRNA injection in 1-cell stage zebrafish eggs. Validation of protein overexpression has been done by western blot. Embryos mortality, developmental delay and morphological abnormalities have been assessed within the first two days of development. Cellular phenotype has been investigated by the analysis of axonal length of 2-days old larvae with confocal microscopy. Motor phenotype of 5-days old larvae has been explored by touched-evoked response assay.Results: The model has been validated by the analysis of well-described ALS mutations, SOD1-Gly93Ala and OPTN Glu478Gly. Overexpression of this mutated protein was shown to provoke a shortening of axons and a premature axonal branching, as well as an impairment of motor performances as expected. We did not observe these aberrations in SOD1-WT injected fishes. Two candidate variants observed in ALS-patients have been explored with our model: SOD1 NM_000454.4:c.400_402del, p.Glu134del and OPTN NM_021980.4:c.1475T > G, p. Leu492Arg. Overexpression of both variants induced morphological abnormalities and motor impairment, suggesting a pathogenic involvement of these variants in ALS-patients.Discussion and conclusions: We developed for the first time a simple animal model, the Zebrafish, useful for the functional analysis of variant pathogenicity in order to assist ALS molecular diagnosis. Our model has been used to assess the pathogenicity of SOD1 and OPTN candidate variants, allowing to improve genetic testing interpretation.

Regulation of Brain Cholesterol: What Role Do Liver X Receptors Play in Neurodegenerative Diseases?

Liver X Receptors (LXR) alpha and beta are two members of nuclear receptor superfamily documented as endogenous cholesterol sensors. Following conversion of cholesterol in oxysterol, both LXR isoforms detect intracellular concentrations and act as transcription factors to promote expression of target genes. Among their numerous physiological roles, they act as central cholesterol-lowering factors. In the central nervous system (CNS), cholesterol has been shown to be an essential determinant of brain function, particularly as a major constituent of myelin and membranes. In the brain, LXRs act as cholesterol central regulators, and, beyond this metabolic function, LXRs have additional roles such as providing neuroprotective effects and lowering neuroinflammation. In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis (MS), dysregulations of cholesterol and oxysterol have been reported. In this paper, we propose to focus on recent advances in the knowledge of the LXRs roles on brain cholesterol and oxysterol homeostasis, neuroinflammation, neuroprotection, and their putative involvement in neurodegenerative disorders. We will discuss their potential use as candidates for both molecular diagnosis and as promising pharmacological targets in the treatment of ALS, AD, or MS patients.

Electrophysiological Characterization of C9ORF72-Associated Amyotrophic Lateral Sclerosis: A Retrospective Study.

OBJECTIVE: C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS). The aim of the present study was to determine whether C9ORF72-associated ALS (C9-ALS) patients present distinctive electrophysiological characteristics that could differentiate them from non C9ORF72-associated ALS (nonC9-ALS) patients. METHODS: Clinical and electrodiagnostic data from C9-ALS patients and nonC9-ALS patients were collected retrospectively. For electroneuromyography, the mean values of motor conduction, myography, and the mean values of sensory conduction were considered. Furthermore, the proportion of ALS patients with electrophysiological sensory neuropathy was determined. RESULTS: No significant difference was observed between 31 C9-ALS patients and 22 nonC9-ALS patients for mean motor conduction and myography. For sensory conduction analyses, mean sensory conduction was not significantly different between both groups. In total, 38% of -C9-ALS patient and 21% of nonC9-ALS patients presented electrophysiological sensory neuropathy (p = 0.33). In -C9-ALS patients with electrophysiological sensory neuropathy, 80% (8/10) were male and 67% (6/9) presented spinal onset compare to 25% (4/16, p = 0.014) male and 25% (4/16, p = 0.087) with spinal onset in those without electrophysiological sensory neuropathy. CONCLUSION: Although not different from nonC9-ALS, these results suggest that sensory involvement is a frequent feature of C9-ALS patients, expanding the phenotype of the disease beyond the motor and cognitive domains.

Parathyroid Hormone Assays following Total Thyroidectomy: Is There a Predictive Value?

OBJECTIVES: Parathyroid hormone (PTH) is a risk marker for hypoparathyroidism (hypoPTH). This study aimed to determine the predictive values of early PTH assays carried out at the moment of skin closure (PTH SC), to establish a treatment algorithm, identifying two threshold values. We assessed the reproducibility of this approach with two different immunoassay kits (hypoPTH) after total thyroidectomy, but its practical application is not consensual. STUDY DESIGN: We conducted a prospective descriptive study, including all patients who underwent a total thyroidectomy between March 2012 and November 2013. Postoperative PTH SC levels, corrected calcium on postoperative days, and occurrence of hypoPTH symptoms were collected. RESULTS: Of 257 patients, the rate of hypoPTH was 20%. Threshold values to obtain a 100% positive predictive value to identify patients for whom hypoPTH was absolutely certain were: PTH SC <7 ng/L for the Roche kit and PTH SC <4 ng/L for the Beckman-Coulter kit. Threshold values to obtain a 100% negative predictive value to identify patients for whom the absence of hypoPTH was absolutely certain were: PTH SC ≥19 ng/L for the Roche kit and PTH SC ≥9 ng/L the Beckman-Coulter kit. CONCLUSIONS: A single serum PTH sampled at skin closure is a reliable test to predict hypoPTH after a total thyroidectomy. The use of a threshold based on a 100% negative predictive value enables patients with no risk of hypoPTH to be safely discharged within the first 24 h postoperatively without unnecessary calcium and vitamin treatment. This medication can be given promptly to patients at risk of hypoPTH to limit the occurrence of hypocalcaemia.